CN101692085A - Method for quantitatively detecting TRAb plate-type fluorescent enzyme immunity and application thereof - Google Patents
Method for quantitatively detecting TRAb plate-type fluorescent enzyme immunity and application thereof Download PDFInfo
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Abstract
The invention relates to a method for quantitatively detecting TRAb plate-type fluorescent enzyme immunity and an application thereof. The method is characterized in that recombination human thyroxin stimulating hormone receptor (hTSHR) extracellular domain amino terminal proteins is taken as antigen to coat a perforated plate and then is sealed by PBST containing 1-5% of BSA; anti-hTSHR first antibody or standard substance, enzyme-labeled second antibody, fluorescent substrate and stopping solution are sequentially added by utilizing the perforated plate as a carrier; and finally fluorescence intensity is measured on a fluorescence illuminant analyzer. The clinical detection of TRAb which mainly comprises TSAb in patient serum has great significance on pathogenic diagnosis, curative effect observation and drug withdrawal occasion determination of GD hyperthyroidism. The invention performs the fluorescence enzyme immunoassay by utilizing a multi-hole site plastic plate as a solid carrier, has easy sheet material source, low cost and easy promotion, and the method is distinctive, sensitive, accurate and stable, thereby being suitable for clinical requirement and promotion.
Description
[technical field]
The invention belongs to medical science ultramicron detection technique field, is to do preparing carriers solid phase antigen ELISA Plate with porous plate, and the board-like luciferase that is used for detection by quantitative human serum TRAb (based on TSAb) is exempted from method (FEIA) and application thereof.
[background technology]
Graves ' sick (GD) is a kind of AITD (AITD) of pilosity, and thyrotropin receptor (TSHR) is a topmost autoantigen in the GD morbidity.TSHR is a kind of glycoprotein that exists on follicular epithelial cell (TEC) film, and it is with after thyroid-stimulating hormone (TSH) combines, and regulates the growth, differentiation of TEC and synthetic and discharge the function of thyroid hormone.On E﹠H factor basis, the body's immunity ANOMALOUS VARIATIONS causes AITD, TSHR is one of main autoantigen of AITD, stimulate body to produce serum thyrotropin receptor antibody (TRAb), this autoantibody belongs to heterogeneous antibody, comprises thyroid stimulating antibody (TSAb) and thyroid gland blocking antibody (TSBAb), and the former simulates the TSH function, stimulate synthetic release of thyroid hormone for a long time constantly and cause GD, the function of appeal is hyperfunction; The latter combines with the TSHR specific site and stops combining and the performance of physiological action of TSH and TSHR, shows as hypothyroidism.At present, TSAb is sure substantially to the pathogenic effects of GD, studies show that in a large number, and the epitope of TSAb mainly is distributed in TSHR film outskirt aminoterminal.TRAb be GD main diseases because of, the morbidity of GD, develop and lapse in play an important role.Detect in the human serum TRAb based on TSAb and be the important indicator on GD etiological diagnosis, observation of curative effect and definite drug withdrawal opportunity.
At present, the homemade TRAb detection technique of being satisfied with without comparison.The external correlation technique of introducing has 2 kinds, and (1) radioreceptor assay (2) enzyme is exempted from method, and its positive rate is all not ideal enough, can not distinguish TSAb and TSBAb, and price is very expensive, and fails at home so far to promote.Also have a kind of biological analysis method can distinguish TSAb and TSBAb abroad, but method is loaded down with trivial details, long flow path and costing an arm and a leg can only be used for scientific research, unsuitable conventional the use.In recent years learn the report of analytic approach again relevant for luminous organism, can be used for detecting TSAb and TSBAb.Be characterized in utilizing Protocols in Molecular Biology to set up the Chinese hamster ovary celI system of TSHR gene and luciferase reporter gene cotransfection and energy stably express, the mensuration of output that excuse me detects the TSAb activity.This method can be distinguished TSAb and TSBAb, but also is Primary Study at present, and technology is prematurity still, can't be used for routine clinical detection.
Human thyrotropin receptor extracellular region aminoterminal gene expression product and preparation method and the application of this product in enzyme immune technology are disclosed among the prior art CN2006100153835.This expressing protein is rich in the antigenic determinant of people TSAb, has the immunocompetence that combines with people TSAb.So the present invention does antigen with reorganization hTSHR film outskirt aminoterminal albumen, with the multi-hole position plastic plate is the luciferase immunological technique of the detection by quantitative people TRAb (based on TSAb) of solid phase carrier, the detection technique of creating the detection TRAb of China oneself and distinguishing to some extent TSAb and TSBAb is very necessary, is numerous clinicians' an urgent demand.
[summary of the invention]
The objective of the invention is to do the antigen coated immobilized enzyme target that on ELISA Plate, prepares, set up board-like solid phase detection by quantitative people TRAb (based on TSAb) FEIA technology with this with reorganization hTSHR film outskirt aminoterminal albumen.
The scheme that the present invention is adopted for achieving the above object is a kind of detection by quantitative TRAb plate-type fluorescent enzyme immunity method of design, it is characterized in that doing antigen coated porous plate with reorganization hTSHR film outskirt aminoterminal albumen, with the PBST sealing that contains 1-5%BSA; And then do carrier with porous plate and add anti-hTSHR first antibody or standard items, enzyme mark second antibody, fluorogenic substrate and stop buffer successively again, on the fluorescence radiation analyser, measure fluorescence intensity at last.
The purposes of detection by quantitative TRAb plate-type fluorescent enzyme immunity method of the present invention, the TRAb luciferase that is used to detect based on TSAb is exempted from method FEIA.The luciferase immunization of detection by quantitative people TRAb (based on TSAb) can be as the important indicator on the etiological diagnosis of clinical GD hyperthyroidism, observation of curative effect and definite drug withdrawal opportunity.
Good effect of the present invention:
1, does antigen coatedly on the solid phase carrier porous plate with reorganization hTSHR film outskirt aminoterminal albumen, and then successfully set up detection by quantitative people TRAb (based on TSAb) FEIA with this.
2, board-like solid phase detection by quantitative people TRAb (based on the TSAb) FEIA of Jian Liing combines enzyme-linked immuno assay ELISA and detection technique of fluorescence, susceptibility and specificity with height, easy to operate, accurate, stable, the detection of a large amount of samples can be carried out simultaneously, and the result can be on fluorescence analyser, read rapidly.And multi-hole position plastic plate convenient sources, cheap, be easy to clinical application.
3, board-like solid phase detection by quantitative people TRAb (based on the TSAb) FEIA of Jian Liing has been used for the clinical detection AITD, etiological diagnosis, observation of curative effect and definite drug withdrawal to the GD hyperthyroidism has important value opportunity especially, to differentiating autoimmunity and non-AITD the important clinical meaning arranged also.
[description of drawings]
Fig. 1 is the TRAb typical curve;
Fig. 2 is hTSHR film outskirt aminoterminal proteantigen and people TRAb and hTPOAb association reaction curve;
Fig. 3 is the parallel laboratory test curve map;
Fig. 4 is the correlation detection curve map;
Fig. 5 is various thyropathy patients serum TRAb (based on a TSAb) positive rate curve map.
Be described in detail with reference to accompanying drawing below in conjunction with embodiments of the invention.
[embodiment]
The board-like solid phase detection by quantitative of the present invention people TRAb (based on TSAb) FEIA does antigen coated porous plate with reorganization hTSHR film outskirt aminoterminal albumen, with the PBST sealing that contains 1-5%BSA; And then do carrier with porous plate and add anti-hTSHR first antibody, enzyme mark second antibody, fluorogenic substrate and stop buffer successively, on the fluorescence radiation analyser, measure fluorescence intensity at last.
The amino acid sequence of the short first shape hormone receptor hTSHR film outskirt aminoterminal albumen of described antigen recombined human is:
Met?Gly?Cys?Ser?Ser?Pro?Pro?Cys?Glu?Cys?His?Gln?Glu?Glu?Asp?Phe 16AA
1 7 13
Arg?Val?Thr?Cys?lys?Asp?Ile?Gln?Arg?Ile?Pro?Ser?Leu?Pro?Pro?Ser 32AA
19 25 31
Thr?Gln?Thr?Leu?lys?Leu?Ile?Glu?Thr?His?Leu?Arg?Thr?Ile?Pro?Ser 48AA
37 43
His?Ala?Phe?Ser?Asn?Leu?Pro?Asn?Ile?Ser?Arg?Ile?Tyr?Val?Ser?Ile 64AA
49 55 61
Asp?Val?Thr?Leu?Gln?Gln?Leu?Glu?Ser?His?Ser?Phe?Tyr?Asn?Leu?Ser 80AA
67 73 79
lys?Val?Thr?His?Ile?Glu?Ile?Arg?Asn?Thr?Arg?Asn?Leu?Thr?Tyr?Ile 96AA
85 91
Asp?Pro?Asp?Ala?Leu?lys?Glu?Leu?Pro?His?Leu?lys?Phe?Leu?Gly?Ile 112AA
97 103 109
Phe?Asn?Thr?Gly?Leu?lys?Met?Phe?Pro?Asp?Leu?Thr?lys?Val?Tyr?Ser 128AA
115 121 127
Thr?Asp?Ile?Phe?Phe?Ile?Leu?Glu?Ile?Thr?Asp?Asn?Pro?Tyr?Met?Thr 144AA
133 139
Ser?Ile?Pro?Val?Asn?Ala?Phe?Gln?Gly?Leu 154AA
145 151
Described porous plate is antigen coated to be: every hole adds 16ng-200ng/100 μ l hTSHR on porous plate, and 4 ℃ are spent the night, and use the PBST rinsing then, again with the PBST confining liquid sealing that contains 1-5%BSA, every hole adds 100 μ l, and 25-37 ℃ of incubation 1 hour used the PBST rinsing again.
Described porous plate is the cellular-plastic panels of position, various varying numbers hole.
The anti-hTSHR first antibody of described adding is: with dilution 1: 50-1: human serum sample or known quantity reference material every holes on porous plate of 200 dilutions add 100 μ l, and 25-37 ℃ of incubation used the PBST rinsing after 1 hour.
Described adding enzyme mark second antibody is: every hole adds 1 on porous plate: 5000-1: the goat anti-human igg 100 μ l of the alkali phosphatase enzyme mark of 25000 dilutions, 25-37 ℃ of incubation used the PBST rinsing after 1 hour, use the PBS rinsing again.
Described adding fluorogenic substrate is: (extension rate is 1: 2-1: 15) 100 μ l, 25-37 ℃ of incubation 1 hour with the tetramethyl phosphoric acid umbelliferone (4-MUP) of 0.01-0.05M Tris-HCL (pH7.8-10.0) damping fluid dilution; At last, every hole adds 0.05-0.15M EDTA stop buffer 50-100 μ l on porous plate, 25-37 ℃ incubation 1-20 minute.
Described rinsing is with PBST 200-300 μ l rinsing 1-3 time.
The present invention can also have following mode in concrete enforcement.
One, the preparation of solid phase antigen ELISA Plate
HTSHR film outskirt aminoterminal albumen is cushioned liquid (0.015M Na with bag
2CO
3, 0.035M NaHCO
3, pH9.6,0.02%NaN
3) being diluted to 16ng/100 μ l-200ng/100 μ l, every hole adds 100 μ l, and blank well adds bag and is cushioned liquid 100 μ l, 4 ℃ of back PBST rinsings of spending the night, with the PBST confining liquid sealing that contains 1-5%BSA, every hole adds 100 μ l again, 25-37 ℃ of incubation 1 hour used the PBST rinsing again.
Two, the foundation of board-like solid phase detection by quantitative people TRAb (based on TSAb) FEIA
(1) board-like solid phase FEIA principle
This technology be with antigen coated on 96 hole polystyrene plates, by the first antibody in the immobilised antigen absorption testing sample, after the flush away unreacted matters, the second antibody and the first antibody that add alkali phosphatase enzyme mark carry out association reaction, after the rinsing, add fluorogenic substrate 4-methyl acid phosphate umbelliferone (4-MUP), 4-MUP is decomposed generation 4-methyl umbelliferone (4-MU) and phosphoric acid by alkaline phosphatase, 4-MU produces the fluorescence of 460nm through the back that excites of 355nm exciting light, the amount of antibody is proportional in this fluorescence intensity and the sample, obtain relative fluorescence unit with Fluoroskan Ascent FL apparatus measures, promptly can calculate the concentration of antibody to be measured in the sample according to typical curve.
(2) exploration of top condition
1. antigen coated amount, an anti-dilutability and the dilution selection of fluorogenic substrate
By intersecting the serial dilution permutation and combination, antigen coated amount is that grouping bag quilt is not waited in 16ng/ hole-200ng/ hole on ELISA Plate; One anti-dilutability is that 1-200 doubly dilutes and do not wait grouping; The fluorogenic substrate dilutability is that 1-15 doubly dilutes and do not wait grouping.Under different condition, measure the relative fluorescence unit (RFU) and the ratio thereof of same TRAb (based on TSAb) positive serum and negative serum.The multiple proportioning of result all can make the negative quality controlled serum ratio of RFU positive quality control serum/RFU greater than more than 2.1 times.Our first-selection antigen coated amount be every hole 100ng/100 μ l, an anti-dilutability is 1: 100, the fluorogenic substrate dilutability is 1: 10.
2. fluorogenic substrate, the selection in stop buffer reaction time
Incubation 30min-120min in 37 ℃ of incubators does not wait grouping with reaction system, observes the variation of different time fluorescence radiation intensity, measures its RFU value respectively.Meanwhile, 5min-60min does not wait behind the adding stop buffer 0.05M EDTA, observes the variation of fluorescence intensity.We are first-selected to add 37 ℃ of incubation 60min behind the fluorogenic substrate, adds behind the stop buffer 5min measurement result on the fluorescence radiation analyser again.
(3) foundation of the preparation of standard items and typical curve
1.TRAb the preparation of standard items
Choose the TRAb positive serum, packing, cold doing ,-30 ℃ keep in Dark Place, standby as the standard items of this chamber preparation.
2. the foundation of typical curve
Typical curve is to carry out 6 calibrates with reference to WHO NIBSC 90/672 reference preparation and in conjunction with clinical definite with the TRAb reference material of this chamber development.The homemade standard items concentration in this chamber is respectively 0.07mIU/L, 0.22mIU/L, 0.72mIU/L, 4.26mIU/L, 9.68mIU/L.With TRAb standard items concentration is horizontal ordinate, is ordinate with its corresponding RFU value, is figure and sets up typical curve on semilogarithmic paper.The results are shown in shown in Figure 1.
(4) board-like solid phase detection by quantitative people TRAb (based on TSAb) FEIA operation steps
1. envelope antigen: hTSHR film outskirt aminoterminal albumen is cushioned liquid (0.015M Na with bag
2CO
3, 0.035M NaHCO
3, pH9.6,0.02%NaN
3) being diluted to 100ng/100ul, every hole adds 100 μ l, and blank well adds bag and is cushioned liquid 100 μ l, and 4 ℃ are spent the night.
2. rinsing: every hole is with 300 μ l PBST rinsings 3 times.
3. sealing: every hole adds 100 μ l confining liquids (PBST that contains 3%BSA), 37 ℃ of incubations 1 hour.
4. rinsing: method is with 2.
5. anti-in conjunction with one: every hole adds the test serum 100 μ l of dilution in 1: 100 (dilution is PBS); Each hole of typical curve adds the standard items 100 μ l of known variable concentrations respectively, 37 ℃ of incubations 1 hour.
6. rinsing: method is with 2.
7. anti-in conjunction with two: every hole adds the goat anti-human igg 100 μ l of the alkali phosphatase enzyme mark of dilution in 1: 20000 (diluting with the PBST that contains 1%BSA), 37 ℃ of incubations 1 hour.
8. rinsing: method is used the PBS rinsing 3 times again with 2.
9. adding fluorogenic substrate: every hole adds the 4-MUP 100 μ l of dilution in 1: 10 (with 10.0 dilutions of 0.1MTris-HCl pH of buffer), 37 ℃ of incubations 1 hour.
10. cessation reaction: every hole adds 0.05M EDTA stop buffer 50 μ l, measures the RFU value behind 37 ℃ of incubation 5min on the fluorescence radiation analyser.
(5) methodology is identified
1. specificity
Bag is by hTSHR film outskirt aminoterminal proteantigen, will contain respectively after 20 times, 50 times, 200 times, 500 times of the serum dilutions of high concentration hTPOAb and people TRAb as anti-mensuration, observes itself and the association reaction situation of intersecting of antigen.
The result shows that hTSHR film outskirt aminoterminal albumen combines with people TRAb specificity, and does not have the association reaction of intersection with hTPOAb, the results are shown in shown in Figure 2.
2. precision
(1) variation within batch: once measure each 6 sample of same positive quality control serum and negative quality controlled serum in the experiment respectively, calculate variation within batch (CV%) and be respectively 4.47% and 11.06%.
(2) batch variation: same positive quality control serum and negative quality controlled serum, METHOD FOR CONTINUOUS DETERMINATION is 6 times in two months, and the CV% value was respectively 7.29% and 17.83% between calculating was criticized.
3. the I measured value of sensitivity: this law TRAb (based on TSAb) is 0.07mIU/L
4. accuracy
Add the TRAb of concentration known, concentration is respectively 8.00mIU/L, and 0.40mIU/L and 0.10mIU/L detect with this law, calculate the recovery of high, medium and low concentration sample respectively, and average recovery rate is 97.46%.The results are shown in Table 1.
Table 1 human serum TRAb determination of recovery rates result
5. viability
GD hyperthyroid patient serum is carried out twice two-fold dilution do parallel experiment, dilution curve and typical curve are parallel relation.As shown in Figure 3.
6. correlativity
This law and import radioreceptor assay TRAb detection kit two methods are examined a collection of different course GD patient serum sample 51 examples altogether, two method testing result significant correlations (r=0.767, p＜0.05).(horizontal ordinate is that this law detects TRAb (based on TSAb) concentration, and ordinate is the concentration that commodity TRAb kit detects TRAb) as shown in Figure 4.
Three, the Preliminary Clinical of board-like solid phase detection by quantitative people TRAb (based on TSAb) FEIA (clinical detection 896 routine blood sample analysis)
(1) human serum TRAb (based on the TSAb) positive is cut determining of limit value
Detect 337 routine healthy people (age 20-30 year, male 167 examples, women 170 examples) serum T RAb, the result represents that with x ± s normal population serum T RAb (based on TSAb) concentration is 0.24 ± 0.11mIU/L, is the positive limit value of cutting of 0.46mIU/L with x+2s concentration.
(2) mensuration of various thyropathy patients serum TRAb (based on TSAb)
1. case is originated
Case is all made a definite diagnosis according to data such as patient's clinical symptoms, sign and laboratory examinations all from the out-patient of General Hospital of Tianjin Medical Univ..Various thyroid disease patient 559 examples, sick (GD) hyperthyroidism first visit of Graves ' patient 183 examples (male 81 examples wherein, woman's 102 examples, 18～70 years old age, 40.6 ± 11.2 years old mean age, the hypermetabolism clinical manifestation is all arranged, the thyroid gland anthorisma, serum thyroid shows high function, TRAb is positive more), 120 examples in the GD treatment (through treatment, most serum FT 3, FT4 have reduced to normally).Hashimoto thyroiditis (HT) companion first low just 112 examples (male 39 examples, women 73 examples, 24～79 years old age, 44.5 ± 13.1 years old mean age, the patient has first to subtract clinical manifestation, and thyromegaly matter is tough, serum thyroid shows low-function state, TMAb and TGAb strong positive).Simplex goiter 22 examples (male 8 examples, women 14 examples, 19～67 years old age, 42.0 ± 13.1 years old mean age), nodular goiter 49 examples (male 28 examples, women 21 examples, 22～60 years old age, 31.0 ± 12.5 years old mean age), thyroid adenoma 21 examples (male 10 examples, woman's 11 examples, 17～68 years old age, 38.7 ± 12.4 years old mean age), subacute thyroiditis 52 examples (male 24 examples, woman's 28 examples, 21～66 years old age, 45.0 ± 9.0 years old mean age).
2. testing result
The mensuration concentration of human serum TRAb (based on TSAb) represents with x ± s, positive rate relatively use chi-square criterion.See Table 2, Fig. 5.
Patient TRAb (based on TSAb) 1.42 ± 0.23mIU/L is just sent out in the GD hyperthyroidism, is significantly higher than normal person (p＜0.01), positive rate 87.61%.TRAb (based on TSAb) 0.62 ± 0.35IU/ml in the GD treatment is significantly higher than normal person (p＜0.05), positive rate 55.07%.
The low patient TRAb 0.71 ± 0.14mIU/L that just sends out of HT companion first is significantly higher than normal person (p＜0.01), positive rate 61.88%.
Simplex goiter 0.25 ± 0.12mIU/L, positive rate are 0.Nodular goiter 0.38 ± 0.13mIU/L, positive rate 3.71%.Thyroid adenoma 0.41 ± 0.08mIU/L, positive rate 5.01%.Subacute thyroiditis 0.40 ± 0.10mIU/L, positive rate are 11.69%.
The GD hyperthyroidism is just sent out group serum T RAb (based on TSAb) positive rate and is higher than low group and other each group (p all＜0.01) just sent out of HT companion first.Simple first is swollen, thyroid adenoma, knot first are swollen, scorching group of methylene and normal human serum TRAb no difference of science of statistics (p＞0.05).
Use that board-like solid phase detection by quantitative serum T RAb (based on TSAb) FEIA detects that the GD hyperthyroidism is just sent out, in the GD treatment, HT companion first lowly just sends out, simplex goiter, nodular goiter, thyroid adenoma and subacute thyroiditis patients serum, positive rate is respectively 87.61%, 55.07%, 61.88%, 0,3.71%, 5.01% and 11.69%, wherein just to send out patient's positive rate the highest in the GD hyperthyroidism, and between other each groups significant difference is arranged, P all＜0.01.
Table 2 people TRAb (based on TSAb) FEIA clinical detection result
Annotate:
*Expression is compared P＜0.01 with the normal person;
■Expression is compared P＜0.05 with the normal person;
▲Each group of expression and other is P＜0.01 relatively
Board-like solid phase detection by quantitative serum T RAb (based on TSAb) FEIA is applied to clinical thyropathy patient, and method is special, sensitive, reliable and stable, and the GD positive rate is higher.Be used for detecting patient's serum and the etiological diagnosis of GD hyperthyroidism, observation of curative effect and definite drug withdrawal all had important value opportunity based on the TRAb of TSAb.To differentiating that autoimmunity and non-AITD also have important clinical significance.
Claims (8)
1. a detection by quantitative TRAb plate-type fluorescent enzyme immunity method is characterized in that doing antigen coated porous plate with the short first shape hormone receptor hTSHR film outskirt aminoterminal albumen of recombined human, with the PBST sealing that contains 1-5%BSA; And then do carrier with porous plate and add anti-hTSHR first antibody or standard items, enzyme mark second antibody, fluorogenic substrate and stop buffer successively again, on the fluorescence radiation analyser, measure fluorescence intensity at last.
2. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1 is characterized in that the amino acid sequence of the short first shape hormone receptor hTSHR film outskirt aminoterminal albumen of described antigen recombined human is:
Met?Gly?Cys?Ser?Ser?Pro?Pro?Cys?Glu?Cys?His?Gln?Glu?Glu?Asp?Phe 16AA
1 7 13
Arg?Val?Thr?Cys?lys?Asp?Ile?Gln?Arg?Ile?Pro?Ser?Leu?Pro?Pro?Ser 32AA
19 25 31
Thr?Gln?Thr?Leu?lys?Leu?Ile?Glu?Thr?His?Leu?Arg?Thr?Ile?Pro?Ser 48AA
37 43
His?Ala?Phe?Ser?Asn?Leu?Pro?Asn?Ile?Ser?Arg?Ile?Tyr?Val?Ser?Ile 64AA
49 55 61
Asp?Val?Thr?Leu?Gln?Gln?Leu?Glu?Ser?His?Ser?Phe?Tyr?Asn?Leu?Ser 80AA
67 73 79
lys?Val?Thr?His?Ile?Glu?Ile?Arg?Asn?Thr?Arg?Asn?Leu?Thr?Tyr?Ile 96AA
85 91
Asp?Pro?Asp?Ala?Leu?lys?Glu?Leu?Pro?His?Leu?lys?Phe?Leu?Gly?Ile?112AA
97 103 109
Phe?Asn?Thr?Gly?Leu?lys?Met?Phe?Pro?Asp?Leu?Thr?lys?Val?Tyr?Ser?128AA
115 121 127
Thr?Asp?Ile?Phe?Phe?Ile?Leu?Glu?Ile?Thr?Asp?Asn?Pro?Tyr?Met?Thr?144AA
133 139
Ser?Ile?Pro?Val?Asn?Ala?Phe?Gln?Gly?Leu 154AA
145 151
3. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1, it is characterized in that described porous plate is antigen coated is: every hole adds 16ng-200ng/100 μ l hTSHR on porous plate, 4 ℃ are spent the night, rinsing then, again with the PBST confining liquid sealing that contains 1-5%BSA, every hole adds 100 μ l, and 25-37 ℃ of incubation 1 hour used the PBST rinsing again.
4. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1, it is characterized in that the anti-hTSHR first antibody of described adding is: with dilution 1: 50-1: human serum sample or known quantity reference material every holes on porous plate of 200 dilutions add 100 μ l, and 25-37 ℃ of incubation used the PBST rinsing after 1 hour.
5. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1, it is characterized in that described adding enzyme mark second antibody is: every hole adds 1 on porous plate: 5000-1: the goat anti-human igg 100 μ l of the alkali phosphatase enzyme mark of 25000 dilutions, 25-37 ℃ of incubation used the PBST rinsing after 1 hour, use the PBS rinsing again.
6. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1, it is characterized in that described adding fluorogenic substrate is: with the Tris-HCL damping fluid of 0.01-0.05M pH7.8-10.0 by 1: 2-1: the tetramethyl phosphoric acid umbelliferone 4-MUP100 μ l of 15 dilution proportion, 25-37 ℃ of incubation 1 hour.
7. detection by quantitative TRAb plate-type fluorescent enzyme immunity method according to claim 1 is characterized in that described adding stop buffer is: every hole adds 0.05-0.15M EDTA stop buffer 50-100 μ l on porous plate, 25-37 ℃ incubation 1-20 minute.
8. the purposes of the described detection by quantitative TRAb plate-type fluorescent enzyme immunity of claim 1 method, the TRAb luciferase that is used to detect based on TSAb is exempted from method FEIA.
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| CN106645689A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof |
| CN106908607A (en) * | 2017-03-01 | 2017-06-30 | 天津市宝坻区人民医院 | The detection method of thyrotrophin receptor antibody |
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| CN109001472A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
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| CN102662068A (en) * | 2012-04-19 | 2012-09-12 | 上海蓝怡科技有限公司 | Tetraiodothyronine magnetic particle fluorescent luminous immunoassay kit and preparation method thereof |
| CN102735850A (en) * | 2012-07-16 | 2012-10-17 | 天津医科大学总医院 | Enzyme-linked immunosorbent assay kit for quantitatively detecting human-serum thyrotrophin receptor antibodies and detecting method |
| CN106645689A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof |
| CN106908607A (en) * | 2017-03-01 | 2017-06-30 | 天津市宝坻区人民医院 | The detection method of thyrotrophin receptor antibody |
| CN108761085A (en) * | 2018-05-24 | 2018-11-06 | 成都医学院 | The enzyme linked immunological kit and detection method of thyrotropin receptor parting antibody can be detected simultaneously |
| CN109001472A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
| CN110579593A (en) * | 2019-09-17 | 2019-12-17 | 郑州安图生物工程股份有限公司 | kit for detecting concentration of stimulated thyroid stimulating hormone receptor antibody |
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